Erratum to: Purification and characterization of iron-cofactored superoxide dismutase from Enteromorpha linza

<正>Erratum to:Chinese Journal of Oceanology and Limnology Vol.31 No.6,P.1190-1195,2013http://dx.doi.org/10.1007/s00343-013-3049-3The supporting grant information on the footnote of the original version of this article needs to be altered and updated.The new information is given below.     

Chemical characteristics and anticoagulant activities of two sulfated polysaccharides from Enteromorpha linza (Chlorophyta)

Two sulfated polysaccharides, designated MP and SP, were extracted from the marine green alga Enteromorpha linza using hot water and then purified using ion-exchange and size-exclusion chromatography. The anticoagulant activities of MP and SP were examined by determination of their activated partial thromboplastin time (APTT), thrombin time (TT) and prothrombin time (PT) using human plasma. Results showed that MP and SP were composed of abundant rhamnose with small amounts of xylose and glucuronic acid, whereas SP also contained a small amount of galactose. Approximate molecular weights of MP and SP were 535 and 502 kDa, respectively. As compared with SP, MP had higher contents of sulfate ester (19.0%) and uronic acid (14.9%). The MP mainly consisted of (1→4)-linked rhamnose residues with partially sulfated groups at the C-3 position, and small amounts of (1→3, 4)-linked rhamnose, (1→2, 4)-linked rhamnose, (1→4)-linked glucuronic acid and (1→4)-linked xylose residues. The SP contained abundant (1→4)-linked rhamnose with minor amounts of (1→3)-linked rhamnose, (1→3, 4)-linked rhamnose, (1→2, 4)-linked rhamnose, (1→4)-linked glucuronic acid, (1→4)-linked xylose, and (1→3)-linked galactose residues. The sulfate groups were mainly located at C-3 of (1→4)-linked rhamnose residues. Both MP and SP, in particular the former, effectively prolonged APTT and TT. This work demonstrates that MP and SP have unique structural characteristics distinct from those of other sulfated polysaccharides from Enteromorpha. The MP is a potential source of anticoagulant, and the difference in anticoagulant activities of the two sulfated polysaccharides is directly linked to the discrepancy of their chemical features.     

Purification and characterization of lectin from humoral fluids of Charybdis feriatus

1 INTRODUCTION Lectins have been regarded as having a puta- tive role in non-self recognition in vertebrate and invertebrate immunities (Arason, 1996; Matsushita, 1996; Olafsen, 1996; Vasta et al., 1999; Wilson, et al., 1999; Marques and Barracco, 2000). …     

Purification and characterization of an alkaline protease from Acetes chinensis

An alkaline protease from Acetes chinensis was purified and characterized in this study. The steps of purification include ammonium sulfate precipitation, ion-exchange chromatography with Q-sepharose Fast Flow, gel filtration chromatography with S300 and the second ion-exchange chromatography with Q-sepharose Fast Flow. The protease was isolated and purified, which was present and active on protein substrates (azocasein and casein). The specific protease activity was 17.15 folds and the recovery was 4.67. The molecular weight of the protease was estimated at 23.2 kD by SDS-PAGE. With azocasein as the susbstrate, the optimal temperature was 55°C and the optimal pH value was 5.5. Ion Ca²⁺ could enhance the proteolytic activity of the protease, while Cu²⁺, EDTA and PMSF could inhibit its activity.     

湛江海域浒苔属Enteromorpha种类的形态与显微结构

用比较解剖学方法观察提江沿海浒苔属（Enteromorpha ）海藻的形态结构,并结合统计分析对其进行 物种鉴定。结果表明,依据藻体分校的有元、数量及分校与主枝直径的大小等形态特征,鉴定的浒苔属j海藻有6种1个变型,分别为缘管浒苔（E. linza）、肠浒苔（ E. intestinalis ）、扁浒苔（ E. compressa ） ,浒苔（E. proliferα）、 条浒苔（E. clathratα）、由浒苔（ E·flexω5α）和肠浒苔宽叶变型（ E. intestinalis f.broadifolium） 0藻体长度由 大到小依次为浒苔、条浒苔、缘管浒苔、肠浒苔、扁浒苔和炀浒苔宽叶变型：细胞呈多角形,藻体的位置不同其细胞大小有所变化,除由浒苔和条浒苔外,其他4种1个变型浒苔的中部细胞最小;所有种类的细胞均有淀粉核,其中肠浒苔、缘管浒苔和扇浒苔仅1个淀粉核,而其他种出现多个淀粉核,尤其条浒苔和曲浒苔数量最多,可达6个以上。定量描述浒苔属6种1个变型的形态特征、内部结构,并发现浒苔属海藻非中空营状的结构特征。     

Purification and Characterization of An Alkaline Protease from Acetes chinensis

An alkaline protease from Acetes chinensis was purified and characterized in this study. The steps of purification include ammonium sulfate precipitation, ion-exchange chromatography with Q-sepharose Fast Flow, gel filtration chromatography with S300 and the second ion-exchange chromatography with Q-sepharose Fast Flow. The protease was isolated and purified, which was present and active on protein substrates （azocasein and casein）. The specific protease activity was 17.15 folds and the recovery was 4.67. The molecular weight of the protease was estimated at 23.2kD by SDS-PAGE. With azocasein as the susbstrate, the optimal temperature was 55℃ and the optimal pH value was 5.5. Ion Ca^2＋ could enhance the proteolytic activity of the protease, while Cu^2＋, EDTA and PMSF could inhibit its activity.     

Purification and characterization of vitellin in mature ovary of marine crab <Emphasis Type="Italic">Charybdis japonica</Emphasis>

Vitellin of mature female marine crab Charybdis japonica was purified by high-performance liquid chromatography （HPLC, DEAE-cellulose-16 anion exchange column）. The apparent molecular mass of the vitellin is 546 ku based on the data of native-PAGE. Under denatured condition （SDS-PAGE）, it was found that vitellin was composed of four polypeptides each at 120, 100, 65, and 55 ku. One disulfide bond was detected in the binding of polypeptide subunits. The purified vitellin, contained 4.47% phosphor and 10.6% polysaccharides, and was identified as glyco-lipo-carotenoprotein, according to the PAGE staining data. The purified vitellin can be used as antigen to raise polyclonal antisera in further application.     

Molecular phylogenetic analysis of attached Ulvaceae species and free-floating Enteromorpha from Qingdao coasts in 2007

Based on the sequence data of the nuclear ribosomal DNA internal transcribed spacer(ITS) 1,5.8 S,and ITS 2,the molecular phylogeny was analyzed on Ulvaceae species collected from Qingdao coasts in summer of 2007,including 15 attached Ulva and Enteromorpha samples from 10 locations and 10 free-floating Enteromorpha samples from seven locations.The result supported the monophyly of all free-floating Enteromorpha samples,implying the unialgal composition of the free-floating Enteromorpha,and the attached Ulvaceae species from Qingdao coasts were grouped into other five clades,suggesting that they were not the biogeographic origin of the free-floating Enteromorpha in that season.     

Purification and Characterization of a Novel Hydrolase That Can Specifically Degrade the Polysaccharide Isolated from Green Seaweed Ulva prolifera

The extracellular polysaccharide hydrolase-producing strain EP-1 was isolated from seawater and identified as Paenibacillus pabuli. Furthermore, a homogeneous extracellular polysaccharide hydrolase from Paenibacillus pabuli EP-1 was purified by combining ion-exchange chromatography and size exclusion chromatography with a purification fold of 90.69 and recovery of 16.23%. Characterization of the purified polysaccharide hydrolase revealed a molecular mass of 38 k Da and optimum activity at 45℃ and pH 6.0. The polysaccharide hydrolase maintained its stability within a wide range of pH(3.0–12.0) and thermal stability when the temperature was below 50℃. The presence of Hg²⁺, Fe²⁺, Mn²⁺, Co²⁺ and SDS notably decreased hydrolase activity, and organic solvents such as formaldehyde, acetone, DMF and acetonitrile completely inhibited hydrolase activity. The purified hydrolase had no activity on agar, carrageenan, gellan gum, sodium alginate, or starch, but effectively hydrolyzed the polysaccharide from Ulva prolifera. The Km and Vmax values of this hydrolase were 43.84 mg m L^-1 and 4.33 mg m L^-1 min^-1, respectively. The sequence analysis with quantitative time-of-flight mass spectrometry indicated that the hydrolase was an endoglucanase.     

Molecular diversity of Enteromorpha from the coast of Yantai: a dual-marker assessment

We collected nine Enteromorpha specimens from the coast of Yantai and evaluated their diversity based on analyses of their ITS （internal transcribed spacer） and 5S rDNA NTS （non-transcribed spacer） sequences. The ITS sequences showed slight nucleotide divergences between Enteromorpha linza and Enteromorpha prolifera. In contrast, multiple highly variable regions were found in the ITS region of Enteromorpha flexuosa. In general, there were more variable sites in the NTS region than in the ITS region in the three species. The variations in 5S rDNA NTS sequences indicated that the molecular diversity of Enteromorpha from the coast of Yantai is very high. However, a phylogenetic tree constructed using 5S rDNA NTS sequence data indicated that genetic differences were not directly related to geographical distribution.     

Pyrolytic characteristics and kinetics of the marine green tide macroalgae, Enteromorpha prolifera

The marine macroalgae Enteromorpha prolifera was one of the main algal genera that occurred in the widespread green tides in Qingdao, China, during the summers of 2007, 2008 and 2010. It is thus a plentiful source of biomass and could be used as a biofuel. In this study, the pyrolytic characteristics and kinetics of E. prolifera were investigated using thermogravimetric analysis (TGA) method. Cornstalk and sawdust were used as comparisons. Pyrolytic characteristics were studied using TG-DTG (thermogravimetry-derivative thermogravimetry) curves. Three stages in the pyrolytic process were determined: dehydration, dramatic weight loss and slow weight loss. E. prolifera was pyrolyzed at a lower initial temperature than the two terrestrial biomass forms. The apparent activation energy values for the three types of biomass were calculated and the mechanism functions were determined using 16 different mechanism functions, frequently used in thermal kinetics analysis. Activation energy values varied with mechanism function and the range of activation energy values for E. prolifera, cornstalk, and sawdust were 25-50 kJ/mol, 60-90 kJ/mol and 120-155 kJ/mol, respectively. This indicates that E. prolifera has low thermal stability for pyrolysis and good combustion characteristics.     

Effect of temperature and irradiance on the growth and reproduction of Enteromorpha prolifera J. Ag. （Chlorophycophyta, Chlorophyceae）

Effect of temperature and irradiance on growth and reproduction of Enteromorpha prolifera that bloomed offshore along the Qingdao coast in summer 2008, was studied. It was showed that E. prolifera propagated mainly asexually with specific growth rate (SGR) of 10.47 at 25°C/40 μmol m-2s-1. Under this condition, gametes with two flagellate formed and released in 5 days. At the beginning of the development, the unicell gamete divided into two cells with heteropolarity, and then the apical cell developed into thalli primordial cells, whereas the basal cell developed into rhizoid primordial cells. In 8-day culture, the monoplast gamete developed into juvenile germling of 240 μm in length. Unreleased gametes can develop directly within the alga body. E. prolifera could either reproduce through lateral branching or fragmenting except apomixis revealed by Microscopic observation. On aged tissue of E. prolifera, although the degraded pigments partially remained in faded algal filaments, numerous vegetative ceils could still divide actively in the algal tissues.     

Purification and characterization of 2-haloacid dehalogenase from marine bacterium Paracoccus sp. DEH99, isolated from marine sponge Hymeniacidon perlevis

2-haloacid dehalogenases constitute a group of dehalogenases which are capable of dehalogenating the halogenated organic compounds. So far, the 2-haloacid dehalogenases have been found in many bacteria, but not in Paracoccus genus. In the present study, one enzyme 2-haloacid dehalogenase(designated as Deh99), induced by DL-2-chloropropionate(DL-2-CPA), was purified from the marine bacterium Paracoccus sp. DEH99, isolated from marine sponge Hymeniacidon perlevis. The enzyme of Deh99 was purified to homogeneity by ammonium sulfate precipitation, ion exchange chromatography(Q-Sepharose HP), and Superdex 200 gel filtration chromatography. The molecular weight of Deh99 was estimated to be 25.0 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE), and 50.0 kDa natively by gel filtration chromatography. The enzyme of Deh99 stereospecifically dehalogenated L-2-CPA to produce D-lactate, with an apparent Michaelis-Menten constant(Km) value of 0.21 mmol L-1 for L-2-CPA. The optimal pH and temperature for Deh99 activity were 10.0 and 40℃, respectively. The enzyme of Deh99 acted on short-carbon-chain 2-haloacids, with the highest activity towards monochloroacetate. The activity of Deh99 was slightly affected by DTT and EDTA, but strongly inhibited by Cu2+ and Zn2+. The enzyme of Deh99 shows unique substrate specificity and inhibitor sensitivities compared to previously characterized 2-haloacid dehalogenases and is the reported one about purified 2-haloacid dehalogenase isolated from the bacteria of Paracoccus genus.     

Purification and Characterization of a Novel Lipase from Antarctic Krill

Lipase from Antarctic krill,with a molecular weight of 71.27kDa,was purified with ammonium sulfate precipitation and a series of chromatographic separations over ion exchange(DEAE)and gel filtration columns(Sephacryl S-100),resulting in 5.2%recovery with a 22.4-fold purification ratio.The optimal pH and temperature for enzyme activity were 8.0 and 45℃,respectively.Purified lipase had Km and Vmax values of 3.27mmolL−1 and 2.4Umg−1,respectively,using p-nitrophenyl laurate as the substrate.Lipase activity was enhanced by adding Ca2+and Mg2+ions in the concentration ranges of 0–0.5mmolL−1 and 0–0.3mmolL−1,respectively,while the activity was inhibited by a further increase in these ion concentrations.Fe3+and Cu2+ions showed obvious inhibitory effects on enzyme activity,and the inhibition rates were 71.8%and 53.3%when the ion concentrations were 0.5mmolL−1.     

Screen and effect analysis of immunostimulants for sea cucumber,Apostichopus japonicus

Immunostimulants may improve disease resistance of aquaculture animals by promoting the nonspecific immunity response of the organisms. Five types of saccharides, including chitosan, yeast polysaccharide, burdock oligosaccharide, seaweed polysaccharide and lentinus edodes polysaccharide, were screened for potential use as immunostimulants by using spectrophotometry. The saccharides were injected into Apostichopus japonicus, a sea cucumber, and the lysozyme and superoxide dismutase (SOD) activities of the coelomic fluid and epidermal slime were monitored in six consecutive days. The results show that the lysozyme activity of the animal's coelomic fluid was significantly stimulated on day 2, day 4 and day 6 after the injection of the saccharides (P<0.05). The effects of chitosan and yeast polysaccharide were the most notable. The lysozyme activity of the epidermal slime was significantly increased by chitosana, yeast polysaccharide, seaweed polysaccharide, and burdock oligosaccharide on day 1 and day 2 after the injection (P<0.05). The SOD activity of the coelomic fluid was significantly promoted by the saccharides on day 2 and day 4 post-injection (P<0.05), while the SOD activity of the epidermal slime increased on day 2. These findings indicate that chitosan and yeast polysaccharide are the most effective immunostimulants and potential healthy anti-disease feedstuff for A. japonicus.     

扁舵鲣抗氧化肽分离纯化及结构鉴定

【目的】从扁舵鲣(Auxis thazard)的木瓜蛋白酶酶解产物中分离鉴定具有较高1,1-二苯基-2-三硝基苯肼(DPPH)自由基清除率的抗氧化肽。【方法】用木瓜蛋白酶水解扁舵鲣,以DPPH自由基清除能力为检测指标,通过超滤、凝胶过滤层析和反向高效液相色谱分离抗氧化肽,再经过超高效液相/三重四级杆飞行时间质谱(UPLC/Xevo G2-XS QTOF)进行结构鉴定。【结果】从酶解产物中获得3种抗氧化肽,其氨基酸序列分别为β-丙氨酸-1-甲基-L-组氨酸(241 u)、Gly-Ala-Gly-Gly-Pro(357 u)和Val-Glu(246 u)。【结论】扁舵鲣的木瓜蛋白酶酶解产物含有抗氧化活性的肽类,可为其抗氧化肽的开发提供理论依据。     

Preparation and characterization of magnetic resin made from chitosan and cerium

In this study,the water-based ferromagnetic fluid and magnetic resin made from chitosan and cerium complex(MRCCC) were successfully prepared by using the chemical co-precipitation technique and by the reversed-phase suspension cross-linking polymerization.MRCCC presented uniform and narrow particle size distribution as determined by the Laser Particles Sizer.The Inductively Coupled Plasma-Atomic Emission Spectrometry(ICP-AES),Fourier transform infrared spectroscopy(FT-IR),differential scanning calorimetry(DSC)and X-ray powder diffraction(XRD)study demonstrated that there were iron and cerium existing in MRCCC.The movement of MRCCC under magnetic field proved its magnetic property.The swelling kinetics in water or solutions with different pH indicated that MRCCC could be applied in solutions with pH greater than 1.0.The ferromagnetic fluid particles were stable in MRCCC soaked in solutions with pH >2.0.In view of these results,MRCCC can be used as material for separation,clarification,adsorption,sustained release and hydrolysis activity.     

Purification of antimicrobial peptide from Antarctic Krill (Euphausia superba) and its function mechanism

The preliminary purification and antimicrobial mechanism of antimicrobial peptide from Antarctic Krill were studied in this paper. The results showed that the molecular weight range of antimicrobial polypeptide (CMCC-1) obtained by cation exchange chromatography was between 245-709D as detected by molecular sieve chromatography, and the minimum inhibition concentration (MIC) of CMCC-1 against Staphylococcus aureus was 5.0 mg mL^?1. The antimicrobial mechanism of CMCC-1 was studied with S. aureus as indicator bacterium. Compared with control group, the results of the experimental group in which S. aureus was treated with CMCC-1 were as follows: 1) CMCC-1 could inhibit cell division at logarithmic phase. 2) The protein and reducing sugar content, and the conductivity of culture medium increased, and the activity of alkaline phosphatase and β-galactosidase could be detected in the culture medium. 3) Observation under scanning electron microscope revealed that somatic morphology became irregular, and then somatic surface became coarse. The cell became much smaller, and most somatic cells gathered. The boundary between cells became dim and finally fused as a whole. 4) Observation under transmission electron microscope showed that the surface of S. aureus became rough and the reproducing ability was restrained. The cell wall became thin and the cytoplasm shrunk. Substances inside cell leaked out, which caused cells death. 5) SDS-PAGE analysis showed that some bands disappeared, and the residual bands became vague. 6) The genomic DNA electrophoresis results showed that the genomic DNA bands of S. aureus were not degraded but the brightness significantly reduced. Thus, it is supposed that CMCC-1 could destroy the cell wall and membrane of S. aureu, increase the cell membrane permeability and the leaking-out of intracellular substances, and thus cause the death of S. aureu.     

Purification and photodynamic bioactivity of phycoerythrin and phycocyanin from Porphyra yezoensis Ueda

Phycoerythrin and phycocyanin were purified from Porphyra yezoensis Ueda with their bioactivity determined in this study. Continuous precipitation with ammonium sulfate at different concentrations （10%, 20%, 40%and 50%） increased the purity （A564：A280） of phycoerythrin to 1.49, 3.92 fold of the raw extract （0.38） and the purity （A615：A280） of phycocyanin to 0.70, 3.33 fold of the raw extract （0.21）. Two more times of chromatography with hydroxylapatites finally made the purity of phycoerythrin and phy-cocyanin reach 5.50, 14.47 fold of the raw extract, and 5.10, 24.29 fold of the raw extract, respectviely. The yield of high purity phycoerythrin and phycocyanin were 0.21%and 0.09%of dried P. yezoensis blade, respectively. The photodynamic cytotoxic ex-periment showed that both phycoerythrin and phycocyanin inhibited the growth of liver tumor cells significantly. It was found that 250 mg L-1 purified phycoerythrin and phycocyanin inhibited the growth of hepatocellular carcinoma cells 24 h after laser-irradiation by 80%and 59%, respectively, and 100 mg L-1 purified phycoerythrin and phycocyanin induced the apoptosis of 31.54%and 32.54%of the cells, respectively, 8 h after photodynamic therapy. Oue findings demonstrated that P. yezoensis can serve as photosensitizer （phycoerythrin and phycocyanin） producer.